Purpose To analyze the reliability and validity of a picture-based questionnaire, the Modified Spinal Function Sort (M-SFS). Methods Sixty-two injured workers with chronic musculoskeletal disorders (MSD) were recruited from two work rehabilitation centers. Internal consistency was assessed by Cronbach’s alpha. Construct validity was tested based on four a priori hypotheses. Structural validity was measured with principal component analysis (PCA). Test–retest reliability and agreement was evaluated using intraclass correlation coefficient (ICC) and measurement error with the limits of agreement (LoA). Results Total score of the M-SFS was 54.4 (SD 16.4) and 56.1 (16.4) for test and retest, respectively. Item distribution showed no ceiling effects. Cronbach’s alpha was 0.94 and 0.95 for test and retest, respectively. PCA showed the presence of four components explaining a total of 74% of the variance. Item communalities were >0.6 in 17 out of 20 items. ICC was 0.90, LoA was ±12.6/16.2 points. The correlations between the M-SFS were 0.89 with the original SFS, 0.49 with the Pain Disability Index, −0.37 and −0.33 with the Numeric Rating Scale for actual pain, −0.52 for selfreported disability due to chronic low back pain, and 0.50, 0.56–0.59 with three distinct lifting tests. No a priori defined hypothesis for construct validity was rejected. Conclusions The M-SFS allows reliable and valid assessment of perceived self-efficacy for work-related tasks and can be recommended for use in patients with chronic MSD. Further research should investigate the proposed M-SFS score of <56 for its predictive validity for non-return to work. According to the Global Burden of Disease Study, musculoskeletal disorders (MSD) are a leading cause of work disability, inflicting substantial burden on society and the individual . Long-teAn efficient way of assessing PSE in patients with MSD is the use of questionnaires. However, questionnaires have important limitations for their use in patients with diverse cultural backgrounds. The first is that the use of questionnaires depends on the literacy and linguistic skills of an evaluee, which may be limited in these patients . The secThe SFS was shown to have good predictive value for work resumption in a multilingual European rehabilitation setting . HoweverThis study was embedded within the usual care for patients with MSD referred for an interdisciplinary inpatient work rehabilitation due to the following reasons: (1) having plateaued with previous medical and rehabilitative care; (2) not regaining full work capacity (WC); (3) exceeding expected healing times. Patient recruitment took place from December 2015 to June 2016 in two different Swiss Rehabilitation centers, in Bellikon (Canton Argovie) and Valens . Inclusion criteria were: Patients with chronic (>3 months), nonspecific MSD, age between 18 and 65, willing to participate in the retest after 2 days and to sign informed consent. Patients were excluded if they were pregnant, reported acute comorbidities , had a medically determined limit in lifting capacity <25 kg or were insufficiently proficient in the German language to understand instructions or read the questions.Ethical approval for this study was granted by the Medical Ethics Committee of the Cantons Aargau and St. Gallen (EK AG 2012/073).A test–retest design was chosen to evaluate the measurement properties of the M-SFS. After checking for eligibility, patients filled out demographic data and various questionnaires, including the M-SFS described in the next paragraph. Retest was performed no earlier than 2 days later than the first test to reduce risk for recall bias. The functional capacity evaluation (FCE) tests were performed after completion of the M-SFS retest.The M-SFS purports to measure PSE to perform work-related tasks. The M-SFS contains 20 drawings with simple written descriptions  measures PSE for work-related tasks that involve the spine . The SFSThe Pain Disability Index (PDI) is a self-report instrument assessing the degree to which chronic pain interferes with various daily activities . The PDISelf-reported disability due to low back pain (LBP) was measured with the Oswestry Disability Index (ODI). The ODI contains 10 items: pain intensity, personal care, lifting, walking, sitting, standing, sleeping, sex life, social life and travelling. Each item includes five statements reflecting different levels of perceived disability ranging from no disability (0) to total disability (5). The total score ranges from 0 to 50, a higher score indicates more severe self-reported disability. The ODI is reliable and has been validated in several languages and settings .Pain intensity was measured with an 11-point numeric rating scale (NRS) ranging from no pain (0) to worst pain (10). The patient was asked to rate their actual pain (“pain now”). The NRS is a commonly used scale with proven reliability and validity in patients with MSD with different levels of literacy .Functional capacity evaluation (FCE) is a standardized battery of functional tests that intend to measure a patient’s safe physical ability for work-related activity . For theNormal distribution was visually assessed using P-P plots and tested with the Kolmogorov–Smirnov and the Shapiro–Wilk tests. Floor and ceiling effects for the M-SFS were considered to be present if more than 15% of participants achieved the lowest or highest possible score of the items .Internal consistency was assessed by item-to-total correlations and Cronbach’s alpha. Optimal consistency for measurements at group level was considered when alpha value was between 0.7 and 0.9. Values <0.7 may be indicative for items measuring different traits, values >0.9 may be indicative for item redundancy .The dimensionality of the 20 M-SFS items was measured using principal component analysis (PCA) with Kaiser normalization and Varimax rotation. An Eigenvalue criterion of 1.0 was used for the analysis. Multidimensionality was assumed since the M-SFS contained new items about activities with tasks including postural tolerance and ambulation.Test–retest reliability was expressed as an intraclass correlation coefficient  (ICC). ICC was interpreted as follows: ICC ≥0.90 is excellent; good when ICC was between 0.75 and 0.90; moderate when ICC was between 0.50 and 0.75; and poor when ICC ≤0.50. ICCs were acceptable when ICC ≥0.75, and the lower boundary of the 95% confidence interval of the ICC ≥0.50 . MeasureFour predefined hypotheses on the strength of the association between M-SFS and the original SFS, pain self-reported disability (ODI), self-reported pain (NRS) and physical performance (FCE tests) are displayed in Table The M-SFS was considered valid when the majority (≥80%) of the a priori hypotheses were not rejected . A signiFrom December 2015 to June 2016, 62 subjects were included based on the inclusion criteria  and 56.1 (SD 16.4) for test and retest, and ranged between 16 and 79, and 14 and 80 for test and retest, respectively. In the test, male participants scored 57.9  and female 55.0 . In the retest, male participants scored 64.0  and female 48.0 .Total score item distribution by gender of the first test is displayed in Fig. PCA with fixed factors showed the presence of four components with initial Eigenvalues exceeding 1, explaining 50.2, 11.5, 6.2, and 5.7%, respectively, with a total of 74% of the variance. The inspection of the scree plot revealed four components. For the interpretation of the components Varimax rotation was executed. The rotated solution revealed the presence of a mixed structure of components showing distinct loadings >0.6 for 17 of 20 items, indicating reasonable evidence for multidimensionality Table . Item 16p = .0.068). Hence LOA were +12.6 (upper limit) and −16.2 (lower limit) points . For the 62 patients in the reliability study, mean M-SFS scores for test and retest were 54.4 , and 56.1 , respectively. Mean difference in M-SFS score between test and retest was 1.7 . Correlations of M-SFS scores during retest were higher correlated with the FCE test-results compared to the first test, ranging from 0.50, 0.56 to 0.59 for lifting low, lifting high and lifting horizontally. Hence, the four hypotheses were not rejected and validity of M-SFS was confirmed.Correlations for first test between the M-SFS and the original SFS were 0.89, and −0.49 with pain-related disability (PDI), and −0.37 and −0.33 with self-reported pain (NRS) for test and retest. Correlation coefficients between the M-SFS and FCE tests for the first test were 0.43, 0.46 and 0.52 for lifting low, lifting high and horizontal lifting, respectively. In patients with low back pain, correlation with self-reported disability (ODI) was −0.52. All correlations were significant (lifting & carrying, activities with bending of the spine, prolonged body postures. These components are in line with the themes that emerged from the previous mixed-method study based on expert opinions, interviews with patients and item analysis from clinical study-data [This study assessed test–retest reliability and validity of the M-SFS in 62 patients with chronic nonspecific MSD in a work-related rehabilitation setting. Men rated their physical capacity on average higher than woman. No floor or ceiling effect of the items was observed. Internal consistency as well as test–retest reliability was high. Limits of agreement of 14.8 suggest that true changes in individuals are above 18.1 of the total score. We found a high correlation with the original SFS , suggestThe lower correlations of the M-SFS with physical performance than previously found of the original SFS can, from our point of view, be explained by several factors. First the time of measurement, which in the current study was at the beginning of the rehabilitation stay while the original SFS was compared with physical performance at the end of rehabilitation. It has formerly been shown that self-reported physical capacity becomes more accurate with repeated testing, which happened in the retest . Second,This study revealed on average a 1.7 higher rated physical capacity at the retest measurement. The highest contribution to this systematic error in measurement results from patients with lower M-SFS scores. These patients showed higher total score variations from test to retest (see also Fig. Guiding rehabilitation of workers with MSD by using the M-SFS may supported for the following reasons: First, the M-SFS attempts to measure PSE with specific, picture-based questions. It has been suggested that the closer a question to actual behavior, the more accurately behavior was predicted . TherefoBesides its strengths, this study has limitations. First, additional studies are needed to compare the M-SFS to other work-related measures including tasks of postural tolerance, ambulation or repetitive bending of the spine. Second, the missing follow-up after rehabilitation prevents assessment of the predictive validity of the M-SFS for return to work. A cutoff score of <100 of the original SFS has shown to be predictive for non-return to work and was achieved by 42% of the responding patients with nonspecific chronic low back pain . The equPerceived self-efficacy for work-related tasks can reliably and validly be assessed with the M-SFS. Measurement properties of the M-SFS were similar to those of the original SFS while time for administration is substantially reduced. Hence, we recommend the use of the modified-version of the SFS. Further research should investigate the properties of the M-SFS with regards to other work-related measures and the proposed M-SFS score of <56 for its predictive validity for non-RTW.


Callithrix jacchus) have recently gained popularity in biomedical research as models of aging research. Basically, they confer advantages from other non-human primates due to their shorter lifespan with onset of appearance of aging at 8 years. Old marmosets present some markers linked to neurodegeneration in the brain such as amyloid beta (Aβ)1-42 and Aβ1-40. However, there are no studies exploring other cellular markers associated with neurodegenerative diseases in this non-human primate. Using immunohistochemistry, we analyzed brains of male adolescent, adult, old, and aged marmosets. We observed accumulation of Aβ1-40 and Aβ1-42 in the cortex of aged subjects. Tau hyperphosphorylation was already detected in the brain of adolescent animals and increased with aging in a more fibrillary form. Microglia activation was also observed in the aging process, while a dystrophic phenotype accumulates in aged subjects. Interestingly, dystrophic microglia contained hyperphosphorylated tau, but active microglia did not. These results support previous findings regarding microglia dysfunctionality in aging and neurodegenerative diseases as Alzheimer’s disease. Further studies should explore the functional consequences of these findings to position this non-human primate as animal model of aging and neurodegeneration.Common marmosets ( Our world population is constantly increasing, and life expectancy in developing countries does as well . Aging iAging in marmosets causes diverse alterations in the CNS , such asIn this study, we aimed to assess the presence of human neurodegenerative markers such as hyperphosphorylation of tau protein and activation of microglia cells. We have detected amyloid plaques in the cortex of aged subjects. An abnormally hyperphosphorylated tau protein was present already in adolescent marmosets, whereas those alterations were more pronounced with aging. Microglia phenotype differs across ages, as adult and old groups had abundant active microglia cells, while dystrophic cells increased significantly in aged animals compared to younger subjects. Importantly, tau hyperphosphorylation and aggregation were present only in dystrophic microglia cells. Thus, the presence of these neurodegenerative markers position marmosets as a potential model of neurodegeneration related to aging.Callithrix jacchus) were housed at the German Primate Center, Göttingen, Germany, under standard conditions complying with the European Union guidelines for the accommodation and care of animals used for experimental and other scientific purposes (2007/526/EC). All animal experiments were performed in accordance with the German Animal Welfare Act, which strictly adheres to the European Union guidelines (EU directive 2010/63/EU) on the use of non-human primates for biomedical research. Experienced veterinarians and caretakers constantly monitored the animals. The experiments were ethically approved by the Lower Saxony State Office for Consumer Protection and Food Safety . Animals did not present neurological disorders or other injuries that can cause trauma to the CNS.Laboratory-bred common marmoset monkeys , two adults (Ad: mean age 5.5 years), five old (O: mean age 11 years), and two aged  were obtained from the medial temporal area – temporal, parietal and entorhinal cortex, and hippocampal formation (Bregma 8.00–0.80 mm according to 2O2 (in PBS) for 10 min to inactivate endogenous peroxidase activity. The following washing steps were performed three times, 10 min each, in 0.2% PBS-triton. To block potential non-specific antibody binding, all sections were incubated in 5% BSA  in PBS for 5 min. Subsequently, sections were incubated with the following antibodies: pTau , AT-100 , Aβ1-40 , Aβ1-42 , Iba-1 , and Alz-50  diluted in 0.2% PBS-triton.All sections were pretreated with formic acid (J.T. Baker) during 15 min and with citrate buffer 20X (Sigma) at 94°C for 10 min, except for sections incubated with anti Iba-1and AT-100. Thereafter, slides were permeabilized with 0.2% Triton X100 in PBS (0.2% PBS-triton) during 20 min. Sections were washed in PBS and incubated in 0.3% H1-40 and Aβ1-42, after washing, sections were incubated with biotinylated secondary antibody  diluted in 0.2% PBS-triton during 2 h at room temperature. Subsequently, the sections were washed and incubated with the avidin-biotin complex  in 0.2% PBS-triton for 2 h, according to the producer’s instructions. Finally, antibody binding was visualized with the chromogen 3,3′-diaminobenzidine  0.025%, with 0.01% H2O2 as a catalytic agent. Control sections were processed without the primary antibody. The sections were then washed, mounted on glass slides and left to dry overnight. Dry sections were cover slipped with mounting medium Entellan (Merck).For immunohistochemistry against tau (Phospho Thr231), AβFor Alz-50 and anti-Iba-1 antibodies, sections were incubated with secondary horseradish peroxidase-conjugated antibodies ; for anti-Iba-1  in 0.2% PBS-triton. Hydrogen peroxide (0.01%) and DAB (0.06%) in 0.2% PBS-triton was used to develop the horseradish peroxidase enzymatic reaction. The enzymatic reaction was stopped with 0.2% PBS-triton and then sections were mounted with Entellan (Merck) as described above.Sections were pretreated with formic acid for 15 min, followed by incubation in citrate buffer 20X at 94°C for 10 min. Sections were permeabilized with 0.2% PBS-triton during 20 min. Thereafter, sections were treated with 5% BSA for 5 min, and incubated with the primary antibodies: AT-100 , anti-Iba-1 , and Alz-50  in the presence of 5% horse serum  during 48 h (4°C). One IgG and one IgM primary antibody was used for double labeling. The sections were then washed with 0.2% PBS-triton, and incubated with secondary antibodies: FITC ; Cy5 ; Cy5 ; Alexa488 , with working dilution 1:500 in all cases, diluted in 0.2% PBS-triton. Control sections were processed without the primary antibody. All sections were co-incubated with DAPI  in 0.2% PBS-triton during 30 min. The sections were then washed, mounted on glass slides. Dry sections were cover slipped with mounting medium VectaShield (Vector Laboratories).Nikon Eclipse 80i light microscope equipped with a Nikon DS-Ri1 camera was used to acquire bright-field images under 20× and 40× objectives, whereas for fluorescent labeling a laser scanning microscopy (Leica TCS-SP8) with argon (488 nm), and helium/neon (543 nm) lasers was used. Both lasers were always used with optimized pinhole diameter. Confocal images were obtained as z-stacks of single optical sections. Stacks of optical sections were superimposed as a single image by using the Leica LAS AF 2.6.0 build 7268 software.2 (area of a single image). We used ImageJ software  to determine the area occupied by tau aggregates. To determine the percentage of area in a determined region, the sum of the areas covered by tau aggregates was divided by the total area, and then, multiplied by 100.The immunoreactivity of phospho Thr231 (pTau231), AT-100 and Alz-50 was quantified as follows: For each subject, two brain slices (at least 800 μm apart) were imaged. Thereafter, from each slice we obtained the following images: 14 images from dorsal hippocampus , eight images from entorhinal cortex (layers VI-III), 14 images from parietal (layers VI-III), and 14 images from temporal cortex (layers VI-III). The total area covered from each region was calculated as the total number of images multiplied by 276360 μm2) was scored in 100 ± 10 images. Based on their morphological characteristics they were classified as: resting , active (hypertrophic soma and ramified cells with extensively thick and branched processes), and dystrophic  were imaged. The number of cells with different morphological state per unit area  and tau conformational changes (Alz50). Differences were considered statistically significant when p ≤ 0.05. Data are presented as means ± SEM.For analysis of single immunohistological markers we used one-way ANOVA, followed by a Tukey’s 1-40 and Aβ1-42 in the cortex of aged animals . A large accumulation of Aβ1-42 in the form of diffuse and compact plaques was found in parenchyma. No Aβ1-42 positive staining was observed in the hippocampal formation. Aβ1-40 was present only in the cortex, mainly around blood vessels as diffuse aggregates; however, it was less abundant than Aβ1-42 . We did not detect Aβ1-40/1-42 positive staining in other ages .Diffuse plaques (without visible fibrillar Aβ or dystrophic neurites) and compact plaques (mature plaques intensely labeled with Aβ) were identified in marmoset brains, according to Using antibodies to detect phosphorylation in relevant residues of the tau aggregation process . Quantification of pTau231-ir in these areas showed significant differences between old and aged subjects versus adult and adolescent groups .First, we aimed to determine whether marmosets present hyperphosphorylated tau protein immunoreactive (ir) labeling in the brain . AdolescFigure 4). Further quantification analysis indicated a significant increase of AT100-ir in old and aged animals, versus those adolescent and adult .AT100 labels a phosphorylation site at residues Thr212 and Ser214 . In the Alz50 binds to two discontinuous sequences of tau, one consisting of residues 5–15 at N-terminus and another in the residues 312–322 of the third microtubule-binding repeat domain . Alz50-iFigures 6A–H). In old subjects, a stronger Alz50-ir was located in cytoplasm mainly in a fibrillary form. Moreover, Alz50-ir was present in neurons with a principal cell-like-morphology, but also in structures that resemble glial cells . Aged individuals had stronger Alz50-ir with cytoplasmic fibrillary inclusions and labeling of dendrites of principal neurons, and a dense Alz50-ir in glia like-cells . Statistical analysis showed a significant increase of Alz50-ir in aged animals compared to younger ones .Our results show Alz50-ir in adolescent and adult subjects as a faint staining mainly in cytoplasmic compartments  and parietal  cortices whereas AT100-ir and Alz50-ir were significantly correlated in hippocampus  and temporal cortex . The slope of the linear regression was in all cases higher for pTau231-ir than for AT100 . Moreover, when tau markers where plotted against age, a similar pattern was observed. PTau231 presented the highest correlation in entorhinal  and parietal  cortices compared to AT100-ir and Alz50-ir. However, in hippocampus and temporal cortex, although pTau231-ir was higher than AT100-ir, correlation analysis indicated a stronger association of AT100-ir  with age .In order to determine the strength of the association of markers of hyperphosphorylation with those related to conformational changes of tau protein, Pearson correlation was used. In all regions analyzed, there was a positive correlation between pTau231/AT100 with Alz-50-ir: pTau231-ir was significantly correlated to Alz50-ir in entorhinal . We then sought to determine the microglia’s phenotype in the dorsal hippocampal region of adolescent, adult, old, and aged individuals. The total number of microglia was similar among all ages . However, the morphological classification indicates an age-dependent phenotype. Resting microglia was the most prominent phenotype across ages, being higher in adolescent animals compared to old and aged subjects . Active microglia presented a more homogeneous presence in all ages, but it was significantly decreased in the aged subjects compared to adolescent, adult, and old animals . Dystrophic microglia revealed the lowest frequency across the ontogeny; however, it was higher in the aged animals compared to adolescent and adult animals .It has been described that microglia are subject to different morphological alterations related to their active state during aging and neurodegeneration . MicroglFigures 4 and 6), we aimed to segregate the presence of Alz50-ir and AT100-ir regarding the microglia’s phenotype. We focused on the dentate gyrus as this area presented a large amount of dystrophic microglia. In the aged animals, AT100 was present in every dystrophic cell as dense inclusions, but active microglia did not show AT100-ir . In addition, Alz50-ir was highly present in dystrophic microglia, while active microglia did not present any labeling .As we detected the presence abnormal tau hyperphosphorylation in structures that resembled glia cells , we sought to determine whether dystrophic microglia in this young animals also contain abnormal tau or whether it was exclusive of aged individuals. In adolescents there were very few dystrophic cells, but those were also positive for AT100; although AT100-ir was more diffuse compared to aged animals. Active and resting microglia did not show AT100-ir .As we observed few dystrophic microglia cells in adolescent and adult marmosets  in humans . In Papio anubis . In AD, the spread of tau pathology from entorhinal cortex to the limbic region (hippocampus) takes several years to progress .In order to better understand the association between the hyperphosphorylation of tau and its conformational changes, we performed a correlation analysis between pTau231/AT100 and Alz-50 for each marmoset . We observed that both markers increase proportionally to each other in most of the brain regions; however, pTau231 showed a better correlation index in the entorhinal and parietal cortices . A recent analysis of 2,332 brains of 1- to 100-year-old individuals, clearly showed that, in humans, hyperphosphorylation of tau commences before puberty or in early adulthood and accumulates along aging until NFTs can be observed , and AT100-ir positive (in the form of inclusions). This pattern of immunoreactivity was observed in both the few dystrophic cells found in adolescent individuals and the large amount of dystrophic cells found in the aged animals. In contrast, active microglia were always negative for Alz50 and AT100 in all ages tested.The mechanisms leading to elevated tau hyperphosphorylation and its aggregation in neurodegenerative diseases still remain unclear. On the one side, neuroinflammation has been implicated in driving hyperphosphorylation and aggregation of tau and neurodegeneration in humans  and variin vitro and in vivo, respectively. Thus, microglia seem to play a protective mechanism in the clearance of tau. Furthermore, a deficiency in the capacity of microglia to internalize and degrade extracellular toxic proteins might be related to a higher incidence of neurodegenerative disease. Among the newly identified AD-risk genes, many are important regulators of innate immunity, including the antigen-presenting and phagocytic and degradative functions of circulating macrophages and microglia (On the other hand, more recent research proposes a neuroprotective role of microglia. Increased intracellular levels of phosphorylated tau could be detrimental to neurons. Thus, tau secretion might be a mechanism by which excess of neuronal tau is removed to prevent toxicity. The spread of tau from cell to cell has been proposed to be one of the mechanisms underlying the progression of tau pathologies , 2014. Ricroglia . An argiicroglia .Promoting the uptake of tau aggregates into microglia could be of great importance in the development of more effective therapies against AD and other tauopathies. In mouse models of tau-dependent neurodegeneration, passive immunization with anti-tau monoclonal antibodies has been shown to reduce age-dependent tau pathology, including NFTs, neurodegeneration and behavioral impairment . FurtherIn the present study, the high amounts of hyperphosphorylated tau observed in the dystrophic microglia of aged marmoset could suggest that long termed tau-phagocytosis triggers the dystrophic process since hyperphosphorylated tau appears early in the adolescence.In summary, marmosets seem to be a valuable prospect species for aging research as they present important hallmarks of human brain aging and neurodegeneration.All animal experiments were approved by the Lower Saxony Federal State Office for Consumer Protection and Food Safety, GermanyJR-C: Substantially contributed in the acquisition, analysis, and interpretation of data for the work; and contributed in the drafting the work. Finally approved the version to be published, and agree to be accountable for all aspects of the work. EF: Substantial contributed in the interpretation of data for the work; revising it critically for important intellectual content. He has given final approval of the version to be published; and agreed to be accountable for all aspects of the work. CP-C: Substantial contributed to the conception, design, and interpretation of data for the work; drafting the work and revising it critically for important intellectual content. She has finally approved the version to be published; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We found that PLN-Dex bound specifically to the surface of activated EC are efficiently internalized by EC and reduced the expression of proinflammatory genes, thus preventing the monocyte adhesion and transmigration to/through activated EC. Given intravenously in mice with acute inflammation, PLN-Dex accumulated at a significant high level in the lungs (compared to nontargeted nanoemulsions) and significantly reduced mRNA expression level of key proinflammatory cytokines such as IL-1β, IL-6, and MCP-1. In conclusion, the newly developed nanoformulation, PLN-Dex, is functional in vitro and in vivo, reducing selectively the endothelium activation and the consequent monocyte infiltration and diminishing significantly the lungs' inflammation, in a mouse model of acute inflammation.Inflammation is a common process associated with numerous vascular pathologies. We hypothesized that targeting the inflamed endothelium by coupling a peptide with high affinity for P-selectin to the surface of dexamethasone-loaded lipid nanoemulsions will highly increase their specific binding to activated endothelial cells (EC) and reduce the cell activation. We developed and characterized dexamethasone-loaded lipid nanoemulsions directed towards P-selectin (PLN-Dex) and monitored their anti-inflammatory effects The vascular endothelium constitutes a cell monolayer lining all the blood vessels and is considered to be the largest paracrine organ in the body . Among mVarious pathological mediators including infectious agents, inflammatory molecules (cytokines/chemokines), reactive oxygen species, different antigens, or physical stress generate activation of EC, transduced by an increased expression of cell adhesion molecules. Thus, EC and their related surface-exposed cell adhesion molecules represent an attractive and accessible target for therapeutic intervention in a myriad of inflammatory diseases.A potential molecular target of EC is P-selectin (CD62P), an inducible cell adhesion molecule overexpressed on the surface of activated endothelium in pathologically altered vasculature . P-SelecPrevious studies showed the feasibility of using P-selectin as a therapeutic target in preclinical studies for different inflammatory pathologies. Thus, the administration of a P-selectin recombinant ligand (rPSGL-Ig) reduced the neointimal hyperplasia after balloon injury, by inhibiting the inflammatory and thrombotic responses in porcine coronary arteries . The samα (TNF-α), interleukin-1 beta (IL-1β), IL-2, and IL-6  , 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine Rhodamine B sulfonyl)  (Rhodamine-PE), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (PEG-DSPE) from Avanti Polar Lipids ; and Dulbecco's modified Eagle's medium (DMEM), RPMI medium, foetal calf serum (FCS), penicillin G, and streptomycin from Gibco BRL ; the cell culture plates were supplied by Corning ; transmigration chambers were from Costar Europe Ltd. . Tris(2-carboxyethyl) phosphine (TCEP) was purchased from Thermo Scientific ; 2′7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was from Life Technologies ; Amicon centrifugal filter columns with a cut-off of 100 kDa were from Millipore. The peptide with high affinity for P-selectin (CGGSSLVSVLDLEPLDAAWL) was synthesized by GeneCust . Deionized (18.2 MΩ/cm) water was generated in-house using a Milli-Q system from Millipore.Reagents were obtained from the following sources: dexamethasone, soybean oil, glycerin, dialysis bag (cut-off size 10 kDa), 3--2,5-diphenyltetrazolium bromide, dexamethasone, lipopolysaccharides fromμM) and Mal-PEG-DSPE (200 μM)), soybean oil (50 μL), and dexamethasone (Dex) (250 μg) dissolved in chloroform, was evaporated using a vacuum rotary evaporator. The aqueous phase containing 1 mL of double distilled water and glycerin (100 μL) was added to the organic phase and sonicated for 5 min in a water bath, at 40 V intensity using a UP200H probe-type sonicator (Heidolph). The lipid nanoemulsions (LN) obtained were further centrifuged using Amicon centrifugal filter units of 100 kDa in order to separate the Dex-entrapped nanoemulsions from free (nonentrapped) Dex and to eliminate any traces of the organic solvent. As control, LN without dexamethasone were produced. For microscopy studies, the LN were fluorescently labeled with Rhodamine-PE added at a 1 mol% ratio.The LN-Dex were prepared employing the ultrasonication method as previously described . Briefly2HPO4, 10 mM NaH2PO4, 2 mM EDTA, 30 mM, and pH: 6.7). Then, the peptide was added  to LN or LN-Dex suspensions and mixed together at room temperature for 6 hours. After this, to saturate the uncoupled maleimide groups, L-cysteine (1 mM) was added for 30 min. As a purification step, the mixture was centrifuged using Amicon centrifugal filter units of 100 kDa in order to separate the nanoemulsions from uncoupled peptide, L-cysteine, and free (nonentrapped) Dex. At this step, the excess glycerin was either filtrated or retained by the ultrafiltration membrane  using region-of-interest (ROI) function of Living Image 4.3.1. software. Then, the tissues were frozen in liquid nitrogen and stored at −80°C until further assays. Tissue homogenates were obtained utilizing a Silent Crusher M homogenizer from Heidolph.The mice (20–25 grams of weight) were givenμL of free dexamethasone  or Dex encapsulated in lipid nanoemulsions  or empty PLN. After 3 hours, similar concentrations of free dexamethasone or nanoemulsions were reinjected into the mice. At 24 hours after the first i.v. administration, the animals were anesthetized, exsanguinated via open heart puncture, and perfused with cold PBS through the left ventricle while the liver was punctured to allow exclusion of the systemic blood. The heart, lung, liver, spleen, kidney, and the brain were harvested, frozen in liquid nitrogen, and stored at −80°C until further assays. Tissue homogenates were obtained utilizing a Silent Crusher M homogenizer from Heidolph. RT-PCR for inflammatory molecules  was performed as described in the previous section. The primers used are indicated in Mice were given i.v. LPS (0.5 mg/kg) and 100  post hoc tests. Differences were considered to be statistically significant at a level of p < 0.05.The results were expressed as mean ± SD and experiments were performed at least in triplicate. The animal studies included three to seven mice/group. Statistical differences were evaluated using Kruskal-Wallis nonparametric independent analysis, followed by Bonferroni, Dunn-Sidak, and Tukeyμg peptide/μmol total lipids . The entrapment efficiency of dexamethasone into lipid nanoemulsions determined by HPLC is 90 ± 2%, corresponding to 22 μg dexamethasone/μmol total lipid. To determine the influence of serum on the size of PLN-Dex, the nanoemulsions were incubated in PBS buffer, pH: 7.4, supplemented with 50% foetal calf serum for 24 hours. In this case, the size of PLN-Dex was 155 ± 4.3 nm, showing that the exposure of PLN-Dex to serum does not change significantly their size. An explanation of the stability of PLN-Dex in serum can be the presence of phospholipidic derivatives of PEG on the surface of nanoemulsions that can impede the massive adsorption of serum protein on the surface and the aggregation of nanoparticles. Also, we tested the effect of time storage at 4°C and found out that the size of PLN-Dex is not modified significantly after 6 months, when an increase of size with 6.95 ± 2.7% is observed. The polydispersity index was between 0.08 and 0.18.The lipid nanoemulsions employed in this study were obtained by a method developed and characterized in a previous study . The phyα activated human EA.hy926 cells. It was found that 70 ± 4% of the activated EC were positive for P-selectin. Therefore, in all further studies, TNF-α activated EA.hy926 cells were employed to assess the binding and uptake of P-selectin targeted nanoemulsion.In preliminary studies, we evaluated, by flow cytometry, the expression of P-selectin on the surface of TNF-α activated EA.hy926 cells was significantly increased in comparison to binding of nontargeted LN  of Rhodamine-PE labeled nontargeted (LN) and P-selectin targeted lipid nanoemulsions (PLN) to TNF-ere used .α activated cells  by TNF- Figures , confirmα activated EA.hy926 cells at 37°C for different time intervals illustrated a gradual increase of the intracellular fluorescence from 30 min to 8 hours  or P-selectin targeted PLN (PLN-Dex) on TNF-analyzed .α activated EA.hy926 cells with empty PLN did not have any significant effect on the mRNA levels of the inflammatory markers investigated, significantly decreased levels were detected in the case of treatments with free Dex or Dex loaded into nanoemulsions. As expected, free Dex significantly reduced the mRNA expression levels of TNF-α, IL-6, IL-8, and RANTES in activated EA.hy926 cells, as compared to TNF-α incubated cells lacking Dex treatment  compared to the monocyte adhesion to nonactivated EA.hy926 cells /(μW/cm2)] using region-of-interest (ROI) function of Living Image 4.3.1. software.In order to induce acute inflammation in mice, the animals were i.v. injected with LPS (0.5 mg/kg), and after 4 hours, the Rhodamine-PE labeled PLN or LN were given intravenously. The mice were sacrificed 1 hour after the administration of nanoemulsions, the vasculature was washed by heart puncture, and the organs were visualized8 and 820.6 × 108, resp.) or with LPS , with no significant differences between the four groups , while in PBS-injected mice the PLN and LN accumulation in the lungs were similar (15.15 × 108 versus 10.4 × 108). For all the other organs analyzed , similar biodistribution was observed for LPS- and PBS-injected mice and no significant differences were observed between PLN and LN, as it can be observed in Figures In accordance with our previous study of LN biodistribution , we obse Figures . Interesβ in the lysates of lungs, liver, kidneys, and the brain of LPS-injected mice, we observed increased IL-1β levels compared to PBS-injected mice, while the highest difference between the two groups was observed in the lungs lysates  or lipid nanoemulsions . After 3 hours, free dexamethasone or lipid nanoemulsions were reinjected into the mice. Anti-inflammatory effects were investigated 24 hours after the LPS administration. The high mRNA levels of the proinflammatory cytokines analyzed  confirmed the strong inflammatory effect induced by LPS in the mice model of acute inflammation , compared to untreated LPS-injected mice  and 100 ammation . The admted mice . The samβ, IL-6, and MCP-1 by 28.3%, 25%, and 27.8%, respectively. This is in accordance with numerous preclinical studies, where it was shown that administration of antibodies or P-selectin targeting moieties reduced important inflammatory markers [Although not significant, the empty P-selectin targeted PLN slightly decreased the mRNA levels of IL-1 markers –15.Given that the endothelium plays a pivotal role in the development and progression of vascular inflammation and by its strategic position is easily accessible for therapeutic agents given intravenously, the EC represent a rational target for pharmacological intervention. We hypothesize that cell adhesion molecule P-selectin is an appropriate molecular target for nanocarriers directed towards surface of activated endothelium because of its high expression on the plasmalemma of activated EC in both acute inflammatory reactions  and chroα activated EA.hy926 cells increased gradually for a time period of 8 hours, confirming the presence of the nanoemulsions inside the cytosol. Since dexamethasone mechanism of action is intracellular by binding to the cytosolic glucocorticoid receptor [For this study, we developed and characterized lipid nanoemulsions functionalized with a peptide with affinity for P-selectin (PLN) as nanocarriers for the anti-inflammatory drug dexamethasone. We report here that P-selectin targeted nanoemulsions bind and are taken up by activated EC line, EA.hy926 cells, at a higher rate compared to nonfunctionalized LN, attesting that the uptake of PLN is specifically mediated by the peptide coupled on the surface of the nanoemulsions. Moreover, PLN internalization by TNF-receptor , the intreceptor , 22. in vivo, like shear stress, targeted moiety, endocytosis, and intracellular trafficking [ in vivo, and future studies need to be done to decipher these important aspects.Indeed, nanoparticles internalization by EC is dependent on many factors which are relevantfficking , 37. Forfficking . Hence, α activated EA.hy926 cells. One of the most important groups of inflammatory mediators are the cytokines, and over time some of them have come to be considered as markers of the inflammatory process. In our study, PLN-Dex have significantly decreased the gene expression of key proinflammatory cytokines  in EA.hy926 cells activated with TNF-α. This results confirm that the encapsulation of dexamethasone in the nanoemulsions maintained the therapeutic characteristic of the drug, which was potentiated by the targeting approach. The data show a similar reduction of mRNA expression levels of the investigated inflammatory proteins  for all Dex formulations. This could be explained by the fact that, in cell culture condition, after 8 hours of incubation, the cells can take up free Dex and nontargeted LN-Dex by an unspecific mechanism and the concentration of Dex delivered intracellularly is enough to produce the observed anti-inflammatory effect. On the other hand, in order to increase the efficiency of targeted delivery of Dex in EC by PLN-Dex, attempts should be made to further increase the specificity of targeting by optimizing the density of peptide with P-selectin affinity on the surface of nanoemulsions. The proinflammatory cytokines analyzed are known to increase endothelium inflammation and promote leukocytes recruitment and transmigration in the subendothelial space, two early key events in the pathogenesis of inflammatory diseases [The anti-inflammatory effects of Dex-loaded nanoemulsions were monitored in TNF-diseases .In further experiments, we tested whether the treatment of activated EA.hy926 cells with dexamethasone-loaded nanoemulsions has a functional involvement in monocytes adhesion and transmigration. Incubation of activated EA.hy926 cells with PLN-Dex significantly reduced both monocytes adhesion and transmigration through endothelial cells monolayer. However, similar concentrations of free dexamethasone reduced only the monocytes adhesion and not the transmigration process, a fact that can be explained by the different time points taken for the investigation . Hence, the therapeutic effect of free Dex in reducing the cell transmigration inflammatory process was time-limited. This results corroborate well with our previous studies showing that this lipid nanoemulsions formulation has a prolonged time release of encapsulated drug, making them appropriate for a prolonged therapeutic effect . in vitro results were further confirmed by in vivo studies, using a mouse model of LPS-induced acute inflammation. The organ biodistribution analysis of administered P-selectin-targeted nanoformulations revealed a significantly higher accumulation in the lungs, as compared to nontargeted nanoemulsions. This suggests that LPS induces the activation of the vascular EC lining the blood vessels in the lungs and increases the expression of P-selectin on the plasma membrane. The pulmonary vasculature consists in principal of an extensive capillary network that exhibits ~30% of the endothelial surface in the body and receives more than 50% of the entire cardiac output [Thec output . As a rec output , 41. intravenous administration of LPS does not increase surface expression of P-selectin in mice platelets [ intravenous LPS (data not shown).Indeed, P-selectin is also expressed by activated platelets which play an important role in the inflammatory process. Interestingly, as it was previously established,latelets –45. One latelets . In accoβ in the lysates of lungs, liver, kidneys, and the brain and found significantly increased levels compared to PBS-injected mice; the highest IL-1β mRNA level was detected in the lungs.In addition, it has been documented that intravenous injection of LPS induces systemic inflammation in all organs but especially in the lungs , 47. To β, IL-6, and MCP-1 in the lungs of mice with acute systemic inflammation, while LN-Dex administration had no significant effect. In preliminary studies, we observed that i.v. administration of 0.5 mg/kg free Dex had no significant therapeutic effect in reducing the proinflammatory cytokines analyzed; hence, we decided to increase the concentration to 1 mg/kg in our study. The administration of free Dex (1 mg/kg) had no significant effect on mRNA levels of investigated cytokines/chemokines, even though the concentration used was double as compared to the concentration of Dex entrapped into PLN-Dex and LN-Dex (0.5 mg/kg). Overall, the in vivo results validate the in vitro data obtained on cultured endothelial cells, showing that P-selectin targeted lipid nanoemulsions loaded with dexamethasone have a significant anti-inflammatory effect by reducing the gene expression of proinflammatory molecules. The anti-inflammatory effect observed in PLN-Dex treated mice can be explained by a combined effect of dexamethasone delivered by nanoemulsions, together with the blocking of P-selectin on the surface of EC by targeted nanoemulsions, previously reported to contribute to reducing the inflammatory process [Intravenous administration of 0.5 mg/kg PLN-Dex had a significantly positive effect in reducing mRNA levels of important proinflammatory cytokines such as IL-1 process , 13. Our process .The approach described in this study may enable future applicability of many potent anti-inflammatory drugs that today do not make it to the clinical trials because of the lack of specificity and the ensuing severe side effects. The local and disease-specific overexpression of P-selectin in inflamed lungs endothelium makes P-selectin an excellent target molecule for site-specific therapy. This strategy may be of clinical relevance for numerous diseases with an inflammatory component where P-selectin is overexpressed, as is the case for atherosclerosis or rheumatoid arthritis. in vitro in activated cultured endothelial cells and in vivo, in the lungs of a mouse model with acute inflammation. This study demonstrates the potential of cell-specific drug delivery targeted to disease-induced molecules on the endothelial cells' surface as a therapeutic strategy for vascular inflammation.The targeted delivery of dexamethasone to activated endothelium by lipid nanoemulsions coupled with a P-selectin-specific peptide as a homing device selectively decreased the expression of proinflammatory genes and reduced the monocytes adhesion and transmigration through endothelial cells monolayer and


Cannabis sativa that presents antipsychotic, anxiolytic, anti-inflammatory, and neuroprotective effects. In Parkinson’s disease patients, CBD is able to attenuate the psychotic symptoms induced by L-DOPA and to improve quality of life. Repeated administration of reserpine in rodents induces motor impairments that are accompanied by cognitive deficits, and has been applied to model both tardive dyskinesia and Parkinson’s disease. The present study investigated whether CBD administration would attenuate reserpine-induced motor and cognitive impairments in rats. Male Wistar rats received four injections of CBD (0.5 or 5 mg/kg) or vehicle (days 2–5). On days 3 and 5, animals received also one injection of 1 mg/kg reserpine or vehicle. Locomotor activity, vacuous chewing movements, and catalepsy were assessed from day 1 to day 7. On days 8 and 9, we evaluated animals’ performance on the plus-maze discriminative avoidance task, for learning/memory assessment. CBD (0.5 and 5 mg/kg) attenuated the increase in catalepsy behavior and in oral movements – but not the decrease in locomotion – induced by reserpine. CBD (0.5 mg/kg) also ameliorated the reserpine-induced memory deficit in the discriminative avoidance task. Our data show that CBD is able to attenuate motor and cognitive impairments induced by reserpine, suggesting the use of this compound in the pharmacotherapy of Parkinson’s disease and tardive dyskinesia.Cannabidiol (CBD) is a non-psychotomimetic compound from  Alterations in the dopaminergic nigrostriatal pathway are linked to important movement disorders, such as Parkinson’s disease and tardive dyskinesia . ParkinsCannabis sativa, being the most abundant after Δ9-tetrahydrocannabinol (Δ9-THC). CBD antagonizes cannabinoid CB1/CB2 receptors agonists and inhibits the reuptake of anandamide, the main endogenous cannabinoid. CBD is also an agonist of the serotonin receptor 5-HT1A and of the vanilloid receptors TRPV1 and TRPV2 (L-nitro-N-arginine (Cannabidiol (CBD) is one of over 60 compounds of nd TRPV2 . CBD prend TRPV2 . In Parknd TRPV2  and imprnd TRPV2 ,b. Moreoarginine . NonetheThe aim of this study was to investigate whether treatment with CBD would attenuate the motor and cognitive impairments induced by repeated administration of reserpine in rats.n = 40), from our own colony, were used. Animals were maintained in groups of five in Plexiglas cages (41 × 34 × 16.5 cm) under controlled environmental conditions  with free access to food and water. The procedures of the present study were approved by the Ethics Committee of Federal University of São Paulo (N° 7798280515), and followed the guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA, and of the Brazilian law for the use of animals in research (Law Number 11.794). All animals were drug-naïve.Three-month-old male Wistar rats  was prepared daily, diluted in saline and 1% tween-80. Reserpine  was diluted in 0.5% glacial acetic acid and distilled water. CBD and its vehicle were administered intraperitoneally. Reserpine and its vehicle were administered subcutaneously. Solutions were given in a volume of 1 ml/kg of body weight.Locomotor activity  was asseCatalepsy behavior  was asseVacuous chewing movements  were assPlus-maze discriminative avoidance task allows the simultaneous evaluation of learning, memory, anxiety, and locomotor activity . The appDistance traveled in the apparatus  and time spent in each arm  were quantified in training and test sessions at 1-minute intervals by an automated activity monitoring system . Percent time in the open arms  in training session was calculated to evaluate anxiety. Percentage of time in the aversive arm (time spent in aversive enclosed arm/time spent in both enclosed arms), assessed minute by minute across the training session, was employed to assess learning. Total time spent in the aversive vs. non-aversive arms in the training session was also used to evaluate learning. Total time spent in the aversive vs. non-aversive arms in the test session was used to evaluate memory.The observers were blind to the animals’ experimental condition.Figure 1).The animals received four injections of CBD (0.5 or 5 mg/kg) or vehicle on days 2–5, and two injections of reserpine (1 mg/kg) or vehicle on days 3 and 5. The drug regimen for the administration of reserpine was based on previous reports that show reserpine-induced oral dyskinesia, catalepsy, and memory impairments , 2004. Tn = 10/group).The experimental groups were: vehicle-vehicle (control group), vehicle-reserpine, CBD 0.5-reserpine, and CBD 5-reserpine  = 3.694; p < 0.05], 6  and 7 . Bonferroni’s test showed that on day 5 (after injection), the group vehicle-reserpine displayed decreased locomotion when compared to the control group (vehicle-vehicle). On day 6, all the reserpine-treated groups displayed diminished locomotor activity when compared to control. On day 7, vehicle-reserpine and CBD 0.5-reserpine groups, but not CBD 5-reserpine, displayed decreased locomotor activity when compared to control. In all comparisons, CBD-treated groups did not differ from vehicle-reserpine group. Treatment with CBD was not able to attenuate the decrease in locomotion induced by reserpine (Table 1).One-way ANOVA revealed effect of treatment on days 5 (after injection) . Bonferroni’s test showed that vehicle-reserpine group displayed increased catalepsy behavior when compared to control group. CBD-treated groups did not differ from control or from vehicle-reserpine group. Treatment with CBD attenuated the reserpine-induced increase in catalepsy .One-way ANOVA revealed effect of treatment on day 7 , 6 , and 7 . Bonferroni’s test showed that vehicle-reserpine group displayed increased vacuous chewing movements when compared to control group. CBD-treated groups did not differ from control or from vehicle-reserpine group. Treatment with CBD attenuated the reserpine-induced increase in oral movements .One-way ANOVA revealed effect of treatment on days 5 (before injection) , but not of treatment or of an interaction between factors. All groups showed a decrease in the time spent in the aversive arm throughout the session . In addition, paired t-test showed that all groups spent more time in the non-aversive enclosed arm than in the aversive enclosed arm: vehicle-vehicle , vehicle-reserpine , CBD 0.5-reserpine , and CBD 5-reserpine  . All groups showed adequate learning of the task.For the percent time spent in the aversive arm in the training session, repeated one-way ANOVA revealed effect of time  and CBD 0.5-reserpine  groups, but not for the vehicle-reserpine and CBD 5-reserpine groups. Treatment with reserpine impaired animals’ retrieval of the discriminative avoidance task, and this effect was prevented by treatment with CBD 0.5, but not with CBD 5 .In the test session, paired F = 6.362; p < 0.001] (Table 2). All groups treated with reserpine displayed diminished locomotor activity when compared to control group, and treatment with CBD and/or reserpine did not alter animals’ anxiety levels.Moreover, in the training session there was no effect of treatment on the percent time spent in the open arms, but treatment affected the distance traveled [The present study showed that CBD administration ameliorates motor and cognitive impairments promoted by reserpine, an animal model for both Parkinson’s disease and tardive dyskinesia. CBD attenuated the reserpine-induced catalepsy behavior, increase in oral movements and memory deficit, but not the decrease in locomotor activity. In addition, treatment with CBD and/or reserpine did not modify animals’ anxiety levels.Reserpine irreversibly blocks the vesicular monoamine transporters 1 and 2 (VMAT-1 and VMAT-2), interfering with the storage of monoamines in synaptic vesicles. As a consequence, it increases the intracellular levels of monoamines and induces monoamine depletion in nerve terminals. The increase in intracellular levels of monoamines enhances their metabolism by the enzyme monoamine oxidase (MAO), increasing oxidative stress  and the effect on catalepsy was only statistically significant on day 7 (48 h after the second injection of reserpine). Also, the effect of reserpine on memory was observed 96 h after the second injection. The time course for the emergence of motor abnormalities and memory impairments suggests a non-acute progressive effect of reserpine. Therefore, this effect fits better to the oxidative damage than to the acute monoamine depletion induced by reserpine.2 receptors), WIN 55,212-2 (a CB1/CB2 agonist), and L-nitro-N-arginine (a non-selective inhibitor of nitric oxide synthase), therefore showing CBD’s potential on inhibiting the catalepsy induced by different mechanisms of action (Cannabidiol (0.5 or 5 mg/kg) attenuated the increase in catalepsy behavior and in oral dyskinesia, which is in accordance with clinical and pre-clinical findings. In humans, CBD is able to improve dystonia . Parkinsf action .Conversely, CBD was not able to attenuate – only to delay – the reserpine-induced decrease in locomotor activity, suggesting it is effective in inhibiting the emergence of some but not all the parkinsonism-like motor abnormalities. This outcome also indicates that the pathophysiological mechanisms related to the reserpine effect on locomotion are distinct from those on catalepsy and oral movements. In fact, while catalepsy behavior and oral movements in rats are linked to the dopaminergic nigrostriatal pathway , locomotReserpine did not alter anxiety-like behavior or learning, but impaired memory in the discriminative avoidance task. These results are in accordance with previous reports using rats and mice . Cogniti9-THC  of the patent “Fluorinated CBD compounds, compositions and uses thereof. Pub. No.: WO/2014/108899. International Application No.: PCT/IL2014/050023”; Def. US no. Reg. 62193296; 29/07/2015; INPI em 19/08/2015 (BR1120150164927). University of São Paulo licensed it to Phytecs Pharm (Resolução USP No. 15.1.130002.1.1). University of São Paulo has an agreement with Prati-Donaduzzi : “Desenvolvimento de um produto farmacêutico contendo canabidiol sintético e comprovação de sua segurança e eficáacia terapêutica na epilepsia, esquizofrenia, doença de Parkinson e transtornos de ansiedade”. JC received a BSPG-Pharm travel Grant award. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications.The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson’s disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated  For example, we have recently shown that adult dopamine neurons of the mesencephalon, which are predominantly affected in human Parkinson’s disease (PD), can be induced to grow new axons following transduction with constitutively active forms of the kinase Akt or the GTPase hRheb.3 These observations provide an experimental basis for a new approach to neurorestoration for PD. However, both of these molecules are known to participate in oncogenesis. Therefore, to administer them intracerebrally under the control of a general mammalian promoter for clinical treatment would carry significant risk. For clinical therapeutics the safety of this approach would be greatly enhanced by restricting the expression of these molecules to the desired cellular targets. In the case of PD, for the treatment of its motor manifestations, the relevant population is the dopamine neurons of the substantia nigra pars compacta (SNpc).The use of cell selective promoters to regulate the expression of foreign genes delivered by viral vectors offers many advantages for both research and clinical applications. In the research setting, for example, cell specific promoters have found numerous uses to enhance the specificity of optogenetic approaches.TH gene was identified by Coker et al. and shown to be sufficient to drive reporter expression with selectivity for a neuronal cell line.4 This sequence was subsequently used to mediate expression in a human neuroblastoma cell line.5 Gardaneh and colleagues demonstrated that a striking synergy can be achieved by use of both 5′ and 3′ flanking sequences of the hTHp.6 A 3.3 kb sequence derived from the 5′ region of the hTHp was found by Kim and colleagues to drive reporter expression in human neuroblastoma cells but not in immature human neural stem cells.7The promoter for tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine biosynthesis, has received extensive prior characterization for achieving selective expression in these neurons. In addition, it provides a number of options for promoter sequences that can be accommodated by the space limitations of an adeno-associated viral (AAV) vector (≤ 4.7 kb). For the purposes of characterizing the human TH promoter (hTHp) sequences that could ultimately be used in gene therapies for human PD, and for transduction of human induced pluripotent stem cells (iPSCs) derived to acquire the dopamine neuron phenotype, we selected three published hTHp sequences that could be accommodated by AAV vector packaging. A 522 bp sequence derived from the 5′ region of the human in vivo context, we evaluated each of these sequences in living mice by AAV-mediated transduction of the SN and control brain regions.The performance of a promoter can be expected to be modified by the cellular context in which it acts. Therefore, since our ultimate goal is to identify promoter sequences that will retain strength and specificity in the An initial assessment of each of the three hTHp constructs at 4–6 weeks following injection of the SN revealed no toxicity to dopaminergic neurons. Sections immunostained for TH with thionin counterstain revealed no evidence of dopamine neuron loss or an inflammatory response (data not shown).9 One subtype lies predominantly in the dorsal tier of the SNpc and expresses calbindin; the other resides in the ventral tier and preferentially expresses class 1 aldehyde dehydrogenase (AHD2).10 Immunofluorescence for these two markers was performed in order to analyze any possible preferential expression by any of the promoter sequences in either of these two subpopulations . For each of the three partial human promoter sequences we were unable to identify any reporter expression in glia .P = 0.004, one-way analysis of variance) , and a statistically significant difference in comparison to hTHp-522-TagRFP-T which had the lowest number of TagRFP-T-positive neurons  . While tIn order to evaluate the sensitivity and specificity of expression in dopamine neurons of the SN, we quantified the total number of TH-positive neurons, TagRFP-T/Nissl-positive neurons and TagRFP-T/Nissl/TH neurons. The three promoter sequences showed only modest levels of sensitivity for detection of dopamine neurons in the SN . The mosin vitro. We demonstrate here that each of these sequences has the ability to mediate reporter expression in dopamine neurons of the SN in vivo. Therefore, they have potential use in clinical applications.Prior studies of these hTHp sequences have been exclusively 11Our primary goal in assessing these sequences was to determine their specificity, in order to ascertain their potential for minimizing expression in nondopaminergic cells, and thereby reducing the likelihood of off-target effects. Achievement of this goal would be an important step forward in the development of Akt and hRheb for use in gene therapy applications in neurorestorative treatment of PD. Given the oncogenic potential of these molecules, we particularly sought to determine the use of these sequences to minimize expression in glial, as they are the predominant cellular source of primary human brain tumors. We have found that none of these sequences induced reporter expression in astrocytes in the SN. Admittedly some of the neuronal specificity achieved may have been due to the known neurotropic properties of AAV vectors. Nevertheless in our own experience with AAV 2/1 injections of the SN, we have occasionally observed glia to be transduced, particularly near the injection site. In addition, for reasons that are not clear, occasional expression is observed in glia for some particular transgenic proteins when the chicken beta actin promoter is used to mediate expression. Therefore, we believe that these hTHp sequences provide an additional margin of safety for avoiding transduction of glial cells. Furthermore, it may be possible in the future to achieve additional specificity of transduction by use of select or modified AAV serotypes.13 so expression of either molecule in adjacent nondopaminergic neurons could have off target effects such as disturbances in motor control due to altered function in these complex basal ganglia circuits. Injections of stratum and cortex with each of the hTHp sequences failed to induce any reporter expression, so in this assessment excellent specificity was achieved. However, surprisingly, within the SN itself, where patterns of expression were assessed by the identification of dopaminergic neurons by immunohistochemistry, specificity appeared to be much less, with 80% or more of the TagRFP-T-positive neurons not expressing TH. There are several possible reasons for this discrepancy. It is well known that immunohistochemistry does not detect all target antigens in tissue sections, due to limitations on the tissue penetration of protein reagents, and in particular to the entry of antibodies into the cell. Thus immunostaining will produce false negatives for the dopaminergic phenotype. In addition, we have observed that transduction of dopamine neurons with transgenes under either the chicken beta actin or the TH promoter can result in a suppression of expression of endogenous proteins, such as TH, used to identify the cell phenotype . These considerations would not however explain the appearance of reporter expression in the SNpr where few dopamine neurons are located. These neurons are predominantly of the gamma-aminobutyric acid-ergic (GABAergic) phenotype. While TH is expressed in some GABAergic neurons of the brain, particularly those in the olfactory bulb,14 it is not known to be expressed in GABAergic neurons of the SNpr. Therefore, the reason why reporter is expressed in nondopaminergic neurons of the SNpr and yet not that of the striatum or cortex is unknown.We also sought to determine if these sequences limited expression among neurons to the dopamine neurons of the SN. Both Akt and hRheb have biologic effects on nondopaminergic neurons,Among these hTHp sequences we did not identify an important difference in specificity. There was a trend for hTHp-522-TagRFP-T to be more specific but this difference was small. hTHp-522-TagRFP-T did however demonstrate a greater sensitivity for the detection of dopaminergic neurons; it successfully expressed reporter in 76% of the TH-positive neurons, whereas hTHp-5,3-TagRFP-T and hTHp-3.3-TagRFP-T achieved expression in only 33 and 40% of instances respectively.in vivo with similar specificity in the SN. Because hTHp-522 as the smallest allows more space in the cassette for transgenes, and offers a higher level of sensitivity for achieving expression in SN dopamine neurons it may be the most useful in practical applications.We conclude that all of these hTHp sequences are capable of mediating expression in dopamine neurons 15 was obtained from Axxora . TagRFP-T DNA was amplified and cloned into an AAV backbone with the chicken β-actin promoter, and a woodchuck postregulatory element (WPRE) at the 3′ end (pBL). Three partial sequences of the hTHp were based on GenBank Acc# AY211521: 522bp (spanning from −491 to +31),4 a combination of 5′-flanking region (spanning from −509 to +1) and a 976 bp segment of 3′-flanking region DNA extending in the 3′ direction from the PolyA signal,6 and 3.3 kb (spanning from −3204 to +3)7 were produced by polymerase chain reaction amplification from human genomic DNA  and subcloned in pGEM-T vector system (Promega).All vectors used for these studies were AAV1 serotype. A plasmid carrying a photostable fluorescent reporter TagRFP-T12 viral genomes (vg)/ml; for AAV hTHp-5,3-TagRFP-T it was 1 × 1012 vg/ml, and for AAV hTHp-3.3-TagRFP-T it was 2 × 1012 vg/ml.The following primers pairs were used: for the 522bp sequence, 5′-AGACACACGGCCTGGAATCT-3′ (forward), 5′-CCTGTGGCGTGGTGGCGTCGGGGGTGGGCATGGCTCAGTGTGGA-3′ (reverse); and for the 3.3 kb sequence,5′-GACCAGTGTCTTGGGAGTTG-3′ (forward), 5′-CATGGCTCAGTGTGGAGGTCCGG-3′ (reverse). For the 5,3 combined promoter the 5′ flanking sequence was created from the 3.3 kb fragment by subcloning of its 3′terminus. Primers for the 3′ flanking region were based on GenBank Acc#NG_008128: 5′-TCACAATAAAAGAAACTGTGGTCT-3′ (forward), 5′-ACACCCCCAGCTGCAACCT-3′ (reverse). The chicken β-actin promoter in pBL-Tag-RFP-T was then replaced by each of these three partial sequences of hTHp to generate three different constructs: hTHp-522-TagRFP-T, hTHp-5,3-TagRFP-T, and hTHp-3.3-TagRFP-T. All nucleotide sequences in the AAV packaging constructs were confirmed before AAV production. AAVs were produced by the University of North Carolina Viral Vector Core . The titer of AAV hTHp-522-TagRFP-T was 5 × 1010, 2 × 109, and 4 × 109 vg. All injection procedures, as described above, were approved by the Columbia University Institutional Animal Care and Use Committee.Adult (8 weeks) males C57BL/6 were obtained from Charles River Laboratories . Mice were anesthetized with ketamine/xylazine solution and placed in a stereotaxic frame  with a mouse adapter. For the injection of the SN the tip of a 5.0 µl syringe needle (26S) was inserted to stereotaxic coordinates AP: −0.35 cm, ML: +0.11 cm, DV: −0.37 cm (relative to bregma); for the striatum AP: +0.09 cm, ML: +0.22 cm, DV: −0.25 cm (relative to bregma); for the cortex AP: +0.09 cm; ML: +0.22 cm; DV: −0.125cm (relative to bregma). Viral vector suspension in a volume of 2.0 µl was injected at 0.1 µl/min over 20 minutes. Thus mice injected with hTHp-522-TagRFP-T, hTHp-5,3-TagRFP-T, and hTHp-3.3-TagRFP-T were injected with 1 × 10Post 6 weeks of AAV injections, mice were deeply anaesthetized and perfused intracardially with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 mol/l phosphate buffer, pH 7.1. The brain was carefully removed and processed for sectioning on either a Vibratome  or cryostat . For Vibratome sectioning the whole brain was postfixed 48 hours in 4% paraformaldehyde in 0.1 mol/l phosphate buffer, pH 7.1. For cryostat sectioning the brain was blocked into forebrain and midbrain regions. The forebrain was postfixed 48 hours; the midbrain was postfixed according to the specific antibody protocol (see below) and then cryoprotected overnight. Cryostat sections were cut after the brain was rapidly frozen by immersion in isopentane on dry ice. All sections were processed free-floating.Coronal cryostat sections containing SN or striatum (30 µm) and horizontal vibratome sections containing SN (50 µm) were immunostained either for TH  or calbindin  or AHD2  or GFAP . The midbrain was postfixed 48 hours for AHD2 and calbindin immunostaining, and 72 hours for GFAP staining. For staining with polyclonal antibodies sections were then treated with goat antirabbit AlexaFluor488. For GFAP staining with a monoclonal, sections were first treated with biotinylated antimouse IgG, followed by streptavidin AlexaFluor488. For some experiments, we performed a fluorescent Nissl counterstain .Images of neurons expressing TagRFP-T and stained for Nissl and TH were acquired by confocal microscopy performed on 50 µm thick horizontal Vibratome sections through the SN. A Z-stack of images  was obtained for SN ventral, SN dorsal, anterior, and posterior regions. The Argon-ion laser at 488 excitation wavelength was used to visualize the TH-positive neurons, the HeNe laser at 561 excitation wavelength to visualize TagRFP-T-positive neurons, and two-photon microscopy at 800 excitation wavelength for Nissl-positive neurons. For each hTHp construct, two mice were examined. From each mouse, two fields (515 × 515 µm) were obtained in the SNpc ventral, one field in the SNpr ventral, two fields in the SNpc dorsal anterior, two fields in the SNpc dorsal posterior, one field in the SNpr dorsal, for a total of eight fields. In each field, all TH-positive neurons, all TagRFP-T/Nissl-positive neurons and all TagRFP-T/Nissl/TH-positive neurons were counted. The percentage of TagRFP-T/Nissl/TH-positive neurons among the total number of TH-positive neurons (sensitivity) and the percentage of TagRFP-T/Nissl/TH-positive neurons among the total number of TagRFP-T/Nissl-positive neurons (specificity) were determined.Images of neurons expressing TagRFP-T and stained for TH were acquired by confocal microscopy performed on 30 µm thick coronal sections through the SN. Images were obtained from each of three rostro-caudal planes of the SN: anterior to the medial terminal (MT) nucleus of the accessory optic tract, containing the MT and posterior to the MT. The Argon-ion laser at 488 excitation wavelength was used to visualize the TH-positive neurons and the HeNe laser at 561 excitation wavelength was used to visualize TagRFP-T-positive neurons. Four mice for each hTHp-5,3-TagRFP-T and hTHp-3.3-TagRFP-T, 12 mice for hTHp-522-TagRFP-T were examined. Seven fields (775 × 775 µm) spanning the entire SN was acquired from each mouse, with each field slightly overlapping adjacent fields. Based on these images the SN and its borders, based on the TH staining were reconstructed by use of Adobe Photoshop. All TagRFP-T-positive neurons were counted in the delineated area. The total number of TagRFP-T-positive neurons within the three SN planes was determined and compared among the three hTHp constructs.post hoc analysis. All statistical analyses were performed using SigmaStat software (version 12) .Differences between the three constructs were analyzed by one-way analysis of variance and Tukey’s Experiments were designed by A.S.R., N.K., and R.E.B. Experiments were performed by A.S.R., T.K., O.Y., and N.K., A.S.R., N.K., and R.E.B. wrote the manuscript.


We examined the Drosophila melanogaster (d) mutant dDAT-G108Q, which leads to a sleepless phenotype in flies harboring this mutation. Molecular dynamics simulations suggested an unstable structure of dDAT-G108Q consistent with a folding defect. This conjecture was verified; heterologously expressed dDAT-G108Q and the human (h) equivalent hDAT-G140Q were retained in the endoplasmic reticulum in a complex with endogenous folding sensors . Incubation of the cells with noribogaine (a DAT ligand selective for the inward-facing state) and/or pifithrin-μ (an HSP70 inhibitor) restored folding of, and hence dopamine transport by, dDAT-G108Q and hDAT-G140Q. The mutated versions of DAT were confined to the cell bodies of the dopaminergic neurons in the fly brain and failed to reach the axonal compartments. Axonal delivery was restored, and sleep time was increased to normal length (from 300 to 1000 min/day) if the dDAT-G108Q-expressing flies were treated with noribogaine and/or pifithrin-μ. Rescuing misfolded versions of DAT by pharmacochaperoning is of therapeutic interest; it may provide opportunities to remedy disorders arising from folding-defective mutants of human DAT and of other related SLC6 transporters.Folding-defective mutants of the human dopamine transporter (DAT) cause a syndrome of infantile dystonia/parkinsonism. Here, we provide a proof-of-principle that the folding deficit is amenable to correction  DAT operates in relay with the cytosolic vesicular monoamine transporter to replenish vesicular neurotransmitter stores and prepare them for subsequent release. DAT is a target of therapeutically relevant (e.g. methylphenidate) and illicit recreational drugs  (–i.e. the translated protein is retained in the endoplasmic reticulum (ER) in an inactive state (The dopamine transporter (DAT)hinones) . Becausehinones) –4. When inones) –. The vasve state , 3.Folding of proteins can be assisted by small molecules. These are thought to act in a manner analogous to proteinaceous chaperones; they bind to folding intermediates and lower the energy barrier for the transition into a trajectory that is conducive for formation of the fully folded state. The pharmacological chaperoning action of these cognate ligands is hence referred to as pharmacochaperoning  or pharm8–Drosophila melanogaster), which had been subjected to random mutagenesis, and linked to a point mutation in the gene encoding Drosophila DAT (dDAT) (108 is located in the first intracellular loop (IL-1). In SERT, IL-1 interacts with the C terminus via a salt bridge to stabilize the folded structure (A phenotype of reduced sleep was identified in fruit flies (T (dDAT) . The mutT (dDAT) . This sutructure . Accordi108 is located in the transmembrane region of dDAT in the interface between transmembrane (TM) helices TM3 and TM12. The dDAT-G108Q mutation introduces a large side chain into the helix interaction motif GXXXG of TM3 at the interface to TM12 (cf.a). We employed molecular dynamics (MD) simulations to examine the structural effect of the large hydrophilic side chain by using the crystal structure of dDAT . The pertinent observations can be summarized as follows: glutamine was not tolerated at position 108; its presence consistently caused a displacement of the second part of TM12 (cf.b and c). Helix TM12 has a conserved proline in the center of the membrane bilayer resulting in a prominent kink, which divides TM12 into two segments. The C-terminal part of TM12 was pushed away from TM3 in all six simulations carrying the dDAT-G108Q mutation. We quantified the movement of the C-terminal part of TM12 by calculating a root mean square displacement (r.m.s.d.) matrix over all 12 MD simulations. The three wild-type simulations and the three reverted wild-type simulations showed small r.m.s.d. values throughout the trajectory and also relative to each other, indicating a stable conformation of TM12 . In contrast, the MD simulations of the dDAT-G108Q mutant and the reverted dDAT-G108Q mutant showed higher r.m.s.d. values with respect to the starting structure, with respect to each other and in particular with respect to the six MD simulations of wild-type dDAT . These movements indicate that TM12 fails to adopt a stable conformation in dDAT-G108Q.GlyTM12 cf.a. We emp108. We quantified this observation by counting the average number of water molecules within 1.0 nm of the Cα atom of residue 108. The average number of waters was twice as high in the dDAT-G108Q mutants as in wild-type DAT (e). The water density was visualized in a slice, which was placed through the peak of the permeating water in DAT-G108Q (g) and the equivalent position of the wild-type dDAT (f). This slice illustrates the higher water density in the vicinity of residue 108 in dDAT-G108Q (cf. f and g).The side chain of glutamine is polar and preferentially hydrated. We observed a local deformation of the membrane next to residue dDAT-G108Q. In addition, five out of six MD simulations showed water permeation through the deformed membrane and hydration of the side chain of Glntype DAT e. The waAT-G108Q g and theype dDAT f. This s108Q cf. , f and g108 of dDAT is conserved among all species examined and is also found in all members of the SLC6 family  (a). This strict conservation suggests an important role, possibly in protein folding. This conjecture was also supported by the molecular dynamics simulations summarized in Fig. 2b) and the Drosophila Schneider 2 cell line . Cell numbers were comparable after transient transfection of plasmids encoding wild-type and mutant transporters indicating that the transient expression of the mutated transporters did not affect cell viability. In contrast to the expression of the wild-type versions of DAT, the expression of the mutant proteins did not result in any measurable [3H]dopamine uptake (b and c). Lysates prepared from stably transfected HEK293 cells only contained the ER resident core-glycosylated dDAT-G108Q, whereas the bulk of wild-type DAT was in the mature glycosylated form . Folding-deficient mutants of SERT are trapped in the ER in complex with the luminal chaperone calnexin and/or the cytosolic chaperone HSP70-1A , whereas the detergent lysates contained comparable amounts of these chaperones . We also visualized the retention of both hDAT-G140Q (b) and dDAT-G108Q (d) in the endoplasmic reticulum by confocal microscopy; the CFP-tagged mutant transporters were co-localized with YFP-tagged calnexin, but they did not reach the cell membrane, which was delineated by staining with trypan blue. In contrast, the corresponding wild-type hDAT (a) and dDAT (c) reached the cell surface. These observations were confirmed by cell surface biotinylation experiments (e).Glyudogene) a. This sAT-G140Q b and dDAAT-G108Q d in the ype hDAT a and dDAand dDAT c reachederiments e.3H]dopamine . In contrast, uptake of [3H]dopamine was detectable in cells that had been incubated with noribogaine (closed circles in b and d). Pifithrin-μ is a cell-permeable small molecule inhibitor of HSP70 (triangles in b and d). Preincubation with either noribogaine or pifithrin-μ also, albeit only moderately, augmented uptake by cells expressing the pertinent wild-type transporters, i.e. hDAT (a) and dDAT (c). In addition, we verified these findings by measuring 2-β-carbomethoxy-3-β-(4-fluorophenyl)-N-[3H]methyltropane binding in intact cells expressing wild-type hDAT or the G140Q mutant (e). Drosophila DATs were not examined, because the affinity of cocaine and its analogs for dDAT is substantially lower than for the human ortholog  was restored by pharmacochaperoning.The plant-derived alkaloid ibogaine binds to the inward-facing conformation of SERT and DAT , 12. Norof HSP70 . The act.e. hDAT a and dDAand dDAT c. In addQ mutant e. Drosoportholog . It is em) in the presence or absence of pifithrin-μ (5 μm). Preincubation with noribogaine resulted in a concentration-dependent increase in [3H]dopamine uptake by the mutant . In the presence of pifithrin-μ , the pharmacochaperoning effect of noribogaine was enhanced. Thus, the two compounds acted in an additive manner. We confirmed that the enhanced [3H]dopamine uptake seen after preincubation with noribogaine and/or pifithrin-μ arose from an increased ER export of dDAT-G108Q by examining the relative proportion of core-glycosylated ER resident and the mature glycosylated form; it is evident from Fig. 5b that cells, which had been pretreated noribogaine or pifithrin-μ, produced a larger amount of mature core glycosylated band of dDAT-G108Q mutant than untreated control cells. Consistent with the data shown in Fig. 5a, the combination of noribogaine and pifithrin-μ produced the largest increase in the mature core glycosylated form.We also examined whether the combination of noribogaine and pifithrin-μ exerted an additive action; HEK293 cells stably expressing dDAT-G108Q were treated with increasing concentrations of noribogaine , but it reduced the relative amount of HSP70-1A and calnexin, which were found in complex with dDAT-G108Q . The combination of noribogaine and pifithrin-μ again resulted in the largest effect on both the total amount of dDAT-G108Q and on complex formation with calnexin and HSP70-1A .Pretreatment of cells with noribogaine and pifithrin-μ is predicted to reduce the association of dDAT-G108Q with calnexin and HSP70-1A, because stalled folding intermediates are chaperoned along the folding trajectory to a stable minimum energy conformation, which can be subsequently exported from the ER. We verified this prediction by immunoprecipitating dDAT-G108Q from cells that had been treated with noribogaine or pifithrin-μ. In fact, either manipulation increased the total amount of dDAT-G108Q, which was recovered by immunoprecipitation  and their axonal projections , which terminate in the dorsal, medial, and ventral portion of the fan-shaped body, respectively . In contrast, YFP-tagged hDAT-G140Q was confined to the PPL1 and PPM3 cell bodies; here, the presynaptic terminals in the fan-shaped body were devoid of any detectable fluorescence . However, detectable amounts of hDAT-G140Q did enter the axonal compartments and reached the presynaptic sites in the fan-shaped body, if the flies were fed noribogaine starting from first instar larval stage until adulthood . Based on this observation, we also explored whether administration of the pharmacochaperone to adult flies sufficed to promote folding and subsequent ER export of hDAT-G140Q. This was the case; in adult flies, which had been exposed to noribogaine via the food for 5 days, YFP-tagged hDAT-G140Q was visualized in the fan-shaped bodies , albeit at substantially lower levels than YFP-tagged wild-hDAT (cf. f and c).The average sleep time of  is 16 h . Althoug mutants , Wu et a , w1118, and Canton S, which have a normal sleep length. Three- to 5-day-old male flies were placed individually into a monitoring system, which allowed for recording their locomotor activity. On day 1 (12-h light/12-h dark cycle) flies were allowed to recover from the CO2 anesthesia. On day 2, the circadian rhythm was entrained by a 12-h light/12-h dark cycle. Subsequently, the flies were switched to a 12-h dark/12-h dark cycle) and observed for 5 additional days. It is evident from Fig. 7A that the difference in activity level of dDAT-deficient fumin flies and of Canton S control flies was magnified, when the animals were on a 12-h dark/12-h dark cycle. This is consistent with the finding that light exposure suppresses the wake-promoting action of dopamine , which were hyperactive, when compared with w1118 flies (red traces). The locomotor activity of dDAT-G108Q flies, which had received increasing amounts of noribogaine via their food, was reduced in a dose-dependent manner; in fact, dDAT-G108Q flies treated with 100 μm noribogaine in the food pellet  were indistinguishable from w1118 . In contrast, neither the locomotor activity of fumin flies nor of w1118 flies was affected by 10–100 μm noribogaine in the food pellet . It is also evident from Fig. 7b that the effect of noribogaine on the activity of dDAT-G108Q flies was maintained over the entire observation period.We verified that the amount of mutant DAT, which reached the axonal territory in the fan-shaped body, sufficed to restore sleep in dDAT-G108Q flies. In these flies the mutant DAT is under the control of the endogenous promoter. Several fly lines were used as reference, including the DAT-deficient eractive , and cn1dopamine . Accordia and b), i.e. the time expected from dDAT-deficient Drosophila (a) or noribogaine (b), an increase in total sleep time was observed. We used the locomotor activity recorded on day 4 to quantify fly sleep, but we stress that similar results were obtained, if the calculation was based on the entire period ranging from days 3 to 5. In contrast, neither pifithrin (c) nor noribogaine (d) affected the very short sleep time of dDAT-deficient fumin flies (c and d). Hence, we rule out that these compounds had an effect on sleep time in the absence of any DAT, which could serve as substrate for pharmacochaperoning. Similarly, we examined cn1bw1 flies, which were originally used for random mutagenesis to create the Zuker collection. Sleep in these cn1bw1 flies is normal (on average about 1000 min). It was also not changed by the administration of noribogaine (f) and pifithrin-μ (e). Similar results were obtained in CantonS and w1118 flies, which were used as additional control . These observations rule out that noribogaine or pifithrin-μ exerted their action by a nonspecific action, i.e. by effects unrelated to their ability to restore the function of dDAT-G108Q. Finally, we also verified that pharmacochaperoning restored sleep in flies harboring hDAT-G140Q (data not shown). The data summarized in in vivo; if the concentration of pifithrin in the food was held constant at 5 μm, which as such elicited only a very modest increase in sleep time, the additional administration of noribogaine further promoted sleeping; pifithrin-μ shifted the dose-response curve of noribogaine to the left .Analogous experiments were carried also out with increasing amounts of pifithrin-μ . Inactivity for 5 min or more was considered as sleep. As shown before , dDAT-G1osophila . If dDATosophila a or noriibogaine b, an incifithrin c nor noribogaine d affectein flies , c and dibogaine f and pifithrin-μ e. Similai.e. sickle cell anemia  Molecular dynamics simulations showed that the G108Q mutation impaired the ability of TM12 to adopt a stable conformation. (ii) In transfected cells, dDAT-G108Q (and its human counterpart) was visualized in the endoplasmic reticulum rather than at the cell surface; accordingly, it failed to mediate cellular uptake of its substrate. Similarly, dDAT-G108Q accumulated as the ER retained core-glycosylated species, whereas the mature complex-glycosylated form was virtually absent. (iii) Large amounts of dDAT-G108Q were trapped in complex with calnexin and HSP70-1A. This association directly reflects stalling of the mutant along the folding trajectory, because calnexin and HSP70-1A act as endogenous folding sensors. (iv) The folding defect of dDAT-G108Q was overcome by incubating the cells with the pharmacochaperone noribogaine or by the HSP70 inhibitor pifithrin-μ . Earlier work indicated that IL1 was important for surface expression of the norepinephrine transporter . This suggested that dDAT-G108Q could, albeit rarely, also reach a point that precludes the entry of destabilizing water. Pharmacochaperoning may work by allowing the folding trajectory to progress through this or a related conformational species.An important role in the structure of the transporter can be inferred from the fact that the glycine residue equivalent to Glynsporter –29. In fsporter –. Glycineormation  acted inormation –32. Recrrmation –, 8, 10. 108Q cf. d. This si.e. the very large number of available ligands available  and [3H]CFT (76 Ci/mmol) were purchased from PerkinElmer Life Sciences. Cell culture media, supplements, and antibiotics were from Invitrogen. Bovine serum albumin (BSA) and CompleteTM protease inhibitor mixture were from Roche Applied Science ; SDS was from BioMol GmbH ; Tris and scintillation mixture (Rotiszint® eco plus) were from Carl Roth GmbH . Anti-GFP antibody (ab290) was from Abcam Plc . Protein A-Sepharose and anti-rabbit IgG1-antibody linked to horseradish peroxidase were from Amersham Biosciences. All other chemicals were of analytical grade. The 0.4% trypan blue stock solution was purchased from Sigma. Pifithrin was purchased from Sigma. Noribogaine was purchased from Cfm Oskar Tropitzsch GmbH .dopamine, at room temperature. Uptake was terminated by rapid wash with ice-cold KRH buffer. For experiments where mK and Vmax values were determined, [3H]dopamine was diluted with unlabeled dopamine to achieve final concentrations of 0.2, 1, 3, 10, 15, and 30 μm. Background uptake was determined in the presence of 30 μm mazindol  that were dissolved in scintillation medium and counted for radioactivity.HEK293 cells transiently expressing wild-type or mutant transporters were incubated with [d-lysine-coated ibidi® glass bottom chambers. After 24 h, cell imaging was performed. Confocal microscopy was done using a Zeiss LSM780 equipped with an argon laser (at 30 milliwatts) and a ×63 oil immersion objective (Zeiss Plan-Neofluar). The plasma membrane was delineated by staining with trypan blue, and ER was outlined by plasmid-driven expression of YFP-calnexin , and collected by centrifugation. Detergent-insoluble material was removed by centrifugation . Equal amounts of protein (2 mg/sample) were incubated for 16 h with 4 μl of anti-GFP antibody (20 μg IgG). Subsequently, pre-equilibrated protein A-Sepharose (20 mg of protein A/sample) was added and incubated at 4 °C for 5 h with gentle rotation. The beads were collected by centrifugation and then washed three times with lysis buffer. Bound proteins were eluted by denaturation in 0.1 ml of sample buffer containing 40 mm dithiothreitol and 1% mercaptoethanol at 45 °C for 30 min. An aliquot from the original cellular lysate was also denatured in sample buffer, and aliquots were resolved by PAGE. Proteins were transferred onto nitrocellulose membranes, which were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TBST). Membranes were incubated with antibodies directed against GFP, calnexin, or HSP70-1A in TBST overnight at 4 °C. Subsequently, membranes were washed in TBST, and horseradish peroxidase-(HRP)-conjugated secondary antibody (1:5000) was applied, and the immunoreactivity was detected by chemiluminescence. Biotinylation of cell surface proteins was done as described previously  and fixed in 2% paraformaldehyde in PBS for 1 h at room temperature. After fixation, brains were washed three times in 0.1% Triton X-100 in PBS for 20 min. Blocking was performed in 10% goat serum for 1 h at room temperature. Brains were then incubated in primary antibody overnight in PBS containing 3% BSA and 0.3% Triton X-100 at 4 °C. The mouse monoclonal antibody nc82  and rabbit polyclonal IgG directed against GFP  were used as primary antibodies. After three 20-min washes with PBS containing 0.1% Triton X-100, the brains were incubated overnight at 4 °C with secondary antibody in PBS containing 0.3% Triton X-100. Alexa Fluor 488 goat anti-rabbit IgG  and Alexa Fluor 647 goat anti-mouse IgG  were used as secondary antibodies. Following incubation with secondary antibody, the brains were washed three times with PBS containing 0.1% Triton X-100 and were mounted using Vectashield® . Images were captured on a Leica SP5II confocal microscope with 20-fold magnification. Z-stack images were scanned at 1-μm section intervals with a resolution of 1024 × 1024 pixels. Images were processed with ImageJ and Adobe Photoshop CS5.1.Drosophila activity monitor system . Eight to 25 flies were used per experimental condition, and the experiments were reproduced in five independent experiments. Flies were individually housed in 5-mm diameter glass tubes, which contained the food pellet with the indicated concentrations of noribogaine and/or pifithrin-μ, which were freshly dissolved in water and mixed with regular food (5% sucrose in 1% agarose). Flies were kept on a 12:12 h light/dark cycle for the first 2 days. From day 3 on, the cycle was shifted to a 12:12 h dark/dark for 5 additional days. Flies received the indicated drugs throughout the study period, which lasted for 7 days. The locomotion activity was constantly monitored for 7 days. Data from the 2nd day of the dark/dark phase was used for calculations. Activity was measured in 1-min bins, and the pySolo software (Flies were entrained to a 12:12 h light/dark cycle. Three- to five-day-old male flies were used for activity monitoring using the DAM2 software  was usedM. F., A. K., T. H., and S. S. designed the experiments and wrote the paper. A. E. K. and S. S. performed and analyzed the experiments shown in Figs. 6